Lyophilized recombinant VWF formulations

ABSTRACT

Long-term stable pharmaceutical formulations of lyophilized recombinant von-Willebrand Factor (rVWF) and methods for making and administering said formulations are described.

PRIORITY

This application is a divisional of U.S. patent application Ser. No.14/939,364, filed Nov. 12, 2015, now U.S. Pat. No. 10,232,022, which isa continuation of U.S. patent application Ser. No. 12/603,064, filedOct. 21, 2009, now abandoned, which claims priority to U.S. ProvisionalApplication No. 61/107,273, filed Oct. 21, 2008, and this application isalso a continuation of U.S. patent application Ser. No. 14/693,078,filed Apr. 22, 2015, which is a continuation of U.S. patent applicationSer. No. 12/342,646, filed Dec. 23, 2008, now abandoned, which claimspriority to U.S. Provisional Application No. 61/017,881, filed Dec. 31,2007, and which claims priority to U.S. Provisional Application No.61/017,418 filed Dec. 28, 2007.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 25, 2019, isnamed 008073_5131US03_SL.txt and is 54,313 bytes in size.

FIELD OF THE INVENTION

Generally, the invention relates to formulations of lyophilizedrecombinant VWF and methods for making a lyophilized compositioncomprising recombinant VWF.

BACKGROUND OF THE INVENTION

Von Willebrand factor (VWF) is a glycoprotein circulating in plasma as aseries of multimers ranging in size from about 500 to 20,000 kD.Multimeric forms of VWF are composed of 250 kD polypeptide subunitslinked together by disulfide bonds. VWF mediates initial plateletadhesion to the sub-endothelium of the damaged vessel wall. Only thelarger multimers exhibit hemostatic activity. It is assumed thatendothelial cells secrete large polymeric forms of VWF and those formsof VWF which have a low molecular weight (low molecular weight VWF)arise from proteolytic cleavage. The multimers having large molecularmasses are stored in the Weibel-Pallade bodies of endothelial cells andliberated upon stimulation.

VWF is synthesized by endothelial cells and megakaryocytes as prepro-VWFthat consists to a large extent of repeated domains. Upon cleavage ofthe signal peptide, pro-VWF dimerizes through disulfide linkages at itsC-terminal region. The dimers serve as protomers for multimerization,which is governed by disulfide linkages between the free end termini.The assembly to multimers is followed by the proteolytic removal of thepropeptide sequence (Leyte et al., Biochem. J. 274 (1990, 257-261).

The primary translation product predicted from the cloned cDNA of VWF isa 2813-residue precursor polypeptide (prepro-VWF). The prepro-VWFconsists of a 22 amino acid signal peptide and a 741 amino acidpropeptide, with the mature VWF comprising 2050 amino acids (Ruggeri Z.A., and Ware, J., FASEB J., 308-316 (1993)).

Defects in VWF are causal to Von Willebrand disease (VWD), which ischaracterized by a more or less pronounced bleeding phenotype. VWD type3 is the most severe form in which VWF is completely missing, and VWDtype 1 relates to a quantitative loss of VWF and its phenotype can bevery mild. VWD type 2 relates to qualitative defects of VWF and can beas severe as VWD type 3. VWD type 2 has many sub forms, some beingassociated with the loss or the decrease of high molecular weightmultimers. Von Willebrand syndrome type 2a (VW-S-2A) is characterized bya loss of both intermediate and large multimers. VWS-2B is characterizedby a loss of highest-molecular-weight multimers. Other diseases anddisorders related to VWF are known in the art.

U.S. Pat. Nos. 6,531,577, 7,166,709, and European Patent Application No.04380188.5, describe plasma-derived VWF formulations. However, inaddition to quantity and purity issues with plasma-derived VWF, there isalso a risk of blood-born pathogens (e.g., viruses and VariantCreutzfeldt-Jakob disease (vCJD). Further, VWF is known to formaggregates during stress conditions.

Thus there exists a need in the art to develop a stable pharmaceuticalformulation comprising recombinant VWF.

SUMMARY OF THE INVENTION

The present invention provides formulations useful for lyophilization ofrecombinant WAIT, resulting in a highly stable pharmaceuticalcomposition. The stable pharmaceutical composition is useful as atherapeutic agent in the treatment of individuals suffering fromdisorders or conditions that can benefit from the administration ofrecombinant VWF.

In one embodiment, a stable lyophilized pharmaceutical formulation of arecombinant von Willebrand Factor (rVWF) is provided comprising. (a) arVWF; (b) one or more buffering agents; (c) one or more amino acids; (d)one or more stabilizing agents; and (e) one or more surfactants; therVWF comprising a polypeptide selected from the group consisting of: a)the amino acid sequence set out in SEQ ID NO: 3; b) a biologicallyactive analog, fragment or variant of a); c) a polypeptide encoded bythe polynucleotide set out in SEQ ID NO: 1; d) a biologically activeanalog, fragment or variant of c); and e) a polypeptide encoded by apolynucleotide that hybridizes to the polynucleotide set out in SEQ IDNO: 1 under moderately stringent hybridization conditions; the buffer iscomprising of a pH buffering agent in a range of about 0.1 mM to about500 nM and the pH is in a range of about 2.0 to about 12.0; the aminoacid is at a concentration of about 1 to about 500 mM; the stabilizingagent is at a concentration of about 0.1 to about 1000 inn and thesurfactant is at a concentration of about 0.01 g/L to about 0.5 g/L.

In another embodiment, the rVWF comprises the amino acid sequence setout in SEQ H) NO: 3. In still another embodiment, the buffering agent isselected from the group consisting of citrate, glycine, histidine,HEPES, Iris and combinations of these agents. In yet another embodiment,the buffering agent is citrate. In various embodiments, the pH is in therange of about 6.0 to about 8.0, about 6.5 to about 7.5, or about 7.3.In another embodiment, the pH is about 7.3.

In another embodiment, the aforementioned amino acid is selected fromthe group consisting of glycine, histidine, proline, serine, alanine andarginine. In another embodiment, the amino acid is at a concentrationrange of about 0.5 mM to about 300 mM. In still another embodiment, theamino acid is glycine at a concentration of about 15 mM.

In one embodiment of the invention, the rVWF comprises the amino acidsequence set out in SEQ ID NO: 3; wherein the buffering agent is citrateand the pH is about 7.3; and wherein the amino acid is glycine at aconcentration of about 15 mM.

In still another embodiment of the invention, the aforementioned one ormore stabilizing agents is selected from the group consisting ofmannitol, lactose, sorbitol, xylitol, sucrose, trehalose, mannose,maltose, lactose; glucose, raffinose, cellobiose, gentiobiose,isomaltose, arabinose, glucosamine, fructose and combinations of thesestabilizing agents. In one embodiment, the stabilizing agents aretrehalose at a concentration of about 10 mM and mannitol at aconcentration of about 20 g/L.

In yet another embodiment of the invention, the aforementionedsurfactant is selected from the group consisting of digitonin, TritonX-100, Triton X-114, TWEEN-20, PATEN-80 and combinations of thesesurfactants. In still another embodiment, the surfactant is TWEEN-80 atabout 0.01 g/L.

In another embodiment of the invention, the rVWF comprises amino acidsequence set out in SEQ. ID NO: 3; wherein the buffering agent iscitrate at a concentration of about 15 at about pH 7.3; wherein theamino acid is glycine at a concentration of about 15 mM; wherein thestabilizing agents are trehalose at a concentration of about 10 g/L andmannitol at a concentration of about 20 g/L.; and wherein the surfactantis TWEEN-80 at about 0.1 g/L.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows ANCOVA analysis of pooled VWF:RCo activity in lotsevaluated for stability (stored at 5° C.±3° C.).

FIG. 2 shows the increase in residual moisture in rVWF FDP stored at 5°C.±3° C.

FIG. 3 shows the increase in residual moisture in rVWF FDP stored at 40°C.±2° C.

DETAILED DESCRIPTION OF THE INVENTION

Definition of Terms

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Singleton, et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOGY (2d ed. 1994); TI-IE CAMBRIDGE DICTIONARY OF SCIENCEAND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger, et al. (eds.), Springer Verlag (1991); and Hale and Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991).

Each publication, patent application, patent, and other reference citedherein is incorporated by reference in its entirety to the extent thatit is not inconsistent with the present disclosure.

It is noted here that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “comprising,” with respect to a peptide compound, means that acompound may include additional amino acids at either or both amino andcarboxy termini of the given sequence. Of course, these additional aminoacids should not significantly interfere with the activity of thecompound. With respect to a composition of the instant invention, theterm “comprising” means that a composition may include additionalcomponents. These additional components should not significantlyinterfere with the activity of the composition.

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter e.g.,but not limited to blood pressure, blood cell count, cholesterol level)or disease state (e.g., but not limited to cancer, autoimmunedisorders).

As used herein the terms “express,” “expressing” and “expression” meanallowing or causing the information in a gene or DNA sequence to becomemanifest, for example, producing a protein by activating the cellularfunctions involved in transcription and translation of a correspondinggene or DNA sequence. A DNA sequence is expressed in or by a cell toform an “expression product” such as a protein. The expression productitself, e.g. the resulting protein, may also be said to be “expressed.”An expression product can be characterized as intracellular,extracellular or secreted. The term “intracellular” means inside a cell.The term “extracellular” means outside a cell, such as a transmembraneprotein. A substance is “secreted” by a cell if it appears insignificant measure outside the cell, from somewhere on or inside thecell.

As used herein a “polypeptide” refers to a polymer composed of aminoacid residues, structural variants, related naturally-occurringstructural variants, and synthetic non-naturally occurring analogsthereof linked via peptide bonds. Synthetic polypeptides are prepared,for example, using an automated polypeptide synthesizer. The term“protein” typically refers to large polypeptides. The term “peptide”typically refers to short polypeptides.

As used herein a “fragment” of a polypeptide is meant to refer to anyportion of a polypeptide or protein smaller than the full-lengthpolypeptide or protein expression product.

As used herein an “analog” refers to any of two or more polypeptidessubstantially similar in structure and having the same biologicalactivity, but can have varying degrees of activity, to either the entiremolecule, or to a fragment thereof. Analogs differ in the composition oftheir amino acid sequences based on one or more mutations involvingsubstitution, deletion, insertion and/or addition of one or more aminoacids for other amino acids. Substitutions can be conservative ornon-conservative based on the physico-chemical or functional relatednessof the amino acid that is being replaced and the amino acid replacingit.

As used herein a “variant” refers to a polypeptide, protein or analogthereof that is modified to comprise additional chemical moieties notnormally a part of the molecule. Such moieties may modulate themolecule's solubility, absorption, biological half-life, etc. Themoieties may alternatively decrease the toxicity of the molecule andeliminate or attenuate any undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences (1980), Procedure for coupling such moieties toa molecule are well known in the art. For example and withoutlimitation, in one aspect the variant is a blood clotting factor havinga chemical modification which confers a longer half-life in vivo to theprotein. In various aspects, polypeptides are modified by glycosylation,pegylation, and/or polysialylation.

Recombinant VWF

The polynucleotide and amino acid sequences of prepro-VWF are set out inSEQ ID NO:1 and SEQ ID NO:2, respectively, and are available at GenBankAccession Nos. NM_000552 and NP_000543, respectively. The amino acidsequence corresponding to the mature VWF protein is set out in SEQ IDNO: 3 (corresponding to amino acids 764-2813 of the full lengthprepro-VWF amino acid sequence).

One form of useful rVWF has at least the property of invivo-stabilizing, e.g. binding, of at least one Factor VIII (FWIII)molecule and having optionally a glycosylation pattern which ispharmacologically acceptable. Specific examples thereof include VWFwithout the A2 domain thus resistant to proteolysis (Lankhof et al.,Thromb. Haemost. 77: 1008-1013, 1997), and a VWF fragment from Val 449to Asn 730 including the glycoprotein lb-binding domain and bindingsites for collagen and heparin (Pieta et al., Biochem. Biophys. Res.Commun. 164: 1339-1347, 1989). The determination of the ability of a VWFto stabilize at least one FVIII molecule is, in one aspect, carried outin VWF-deficient mammals according to methods known in the state in theart.

The rVWF of the present invention is produced by any method known in theart. One specific example is disclosed in WO86/06096 published on Oct.23, 1986 and U.S. patent application Ser. No. 07/559,509, filed on Jul.23, 1990, which is incorporated herein by reference with respect to themethods of producing recombinant VWF. Thus, methods are known in the artfor (i) the production of recombinant DNA by genetic engineering, e.g.via reverse transcription of RNA and/or amplification of DNA, (ii)introducing recombinant DNA into procaryotic or eucaryotic cells bytransfection, e.g. via electroporation or microinjection, (iii)cultivating the transformed cells, e.g. in a continuous or batchwisemanner, (iv) expressing VWF, e.g. constitutively or upon induction, and(v) isolating the VWF, e.g. from the culture medium or by harvesting thetransformed cells, in order to (vi) obtain purified rVWF, e.g. via anionexchange chromatography or affinity chromatography. A recombinant VWFis, in one aspect, made in transformed host cells using recombinant DNAtechniques well known in the art. For instance, sequences coding for thepolypeptide could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule is, in another aspect,synthesized using chemical synthesis techniques, such as thephosphoramidate method. Also, in still another aspect, a combination ofthese techniques is used.

The invention also provides vectors encoding polypeptides of theinvention in an appropriate host. The vector comprises thepolynucleotide that encodes the polypeptide operatively linked toappropriate expression control sequences. Methods of effecting thisoperative linking, either before or after the polynucleotide is insertedinto the vector, are well known. Expression control sequences includepromoters, activators, enhancers, operators, ribosomal binding sites,start signals, stop signals, cap signals, polyadenylation signals, andother signals involved with the control of transcription or translation.The resulting vector having the polynucleotide therein is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells are used inthe practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art, including, forexample, compatibility with the chosen expression vector, toxicity ofthe peptides encoded by the DNA molecule, rate of transformation, easeof recover of the peptides, expression characteristics, bio-safety andcosts. A balance of these factors must be struck with the understandingthat not all host cells are equally effective for the expression of aparticular DNA sequence. Within these general guidelines, usefulmicrobial host cells include, without limitation, bacteria, yeast andother fungi, insects, plants, mammalian (including human) cells inculture, or other hosts known in the art.

Transformed host cells are cultured under conventional fermentationconditions so that the desired compounds are expressed. Suchfermentation conditions are well known in the art. Finally, thepolypeptides are purified from culture media or the host cellsthemselves by methods well known in the art.

Depending on the host cell utilized to express a compound of theinvention, carbohydrate (oligosaccharide) groups are optionally attachedto sites that are known to be glycosylation sites in proteins.Generally. O-linked oligosaccharides are attached to serine (Ser) orthreonine (Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids not countingproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both N-linked and O-linked oligosaccharides isN-acetylneuraminic acid (referred to as sialic acid). Sialic acid isusually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, in one aspect,confers acidic properties to the glycosylated compound. Such site(s) maybe incorporated in the linker of the compounds of this invention and arepreferably glycosylated by a cell during recombinant production of thepolypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS).In other aspects, such sites are glycosylated by synthetic orsemi-synthetic procedures known in the art.

Alternatively, the compounds are made by synthetic methods using, forexample, solid phase synthesis techniques. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149 Davis et al. (1985),Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins(3rd ed.) 2: 257-527. Solid phase synthesis is the preferred techniqueof making individual peptides since it is the most cost-effective methodof making small peptides.

Fragments, Variants and Analogs of VWF

Methods for preparing polypeptide fragments, variants or analogs arewell-known in the art.

Fragments of a polypeptide are prepared using, without limitation,enzymatic cleavage (e.g., trypsin, chymotrypsin) and also usingrecombinant means to generate a polypeptide fragments having a specificamino acid sequence. Polypeptide fragments may be generated comprising aregion of the protein having a particular activity, such as amultimerization domain or any other identifiable VWF domain known in theart.

Methods of making polypeptide analogs are also well-known. Amino acidsequence analogs of a polypeptide can be substitutional, insertional,addition or deletion analogs, Deletion analogs, including fragments of apolypeptide, lack one or more residues of the native protein which arenot essential for function or immunogenic activity. Insertional analogsinvolve the addition of, e.g., amino acid(s) at a non-terminal point inthe polypeptide. This analog may include, for example and withoutlimitation, insertion of an immunoreactive epitope or simply a singleresidue. Addition analogs, including fragments of a polypeptide, includethe addition of one or more amino acids at either or both termini of aprotein and include, for example, fusion proteins. Combinations of theaforementioned analogs are also contemplated.

Substitutional analogs typically exchange one amino acid of thewild-type for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptidewithout the complete loss of other functions or properties. In oneaspect, substitutions are conservative substitutions. “Conservativeamino acid substitution” is substitution of an amino acid with an aminoacid having a side chain or a similar chemical character. Similar aminoacids for making conservative substitutions include those having anacidic side chain (glutamic acid, aspartic acid); a basic side chain(arginine, lysine, histidine); a polar amide side chain (glutamine,asparagine); a hydrophobic, aliphatic side chain (leucine, isoleucine,valine, alanine, glycine); an aromatic side chain (phenylalanine,tryptophan, tyrosine); a small side chain (glycine, alanine, serine,threonine, methionine): or an aliphatic hydroxyl side chain (serine,threonine).

In one aspect, analogs are substantially homologous or substantiallyidentical to the recombinant VWF from which they are derived. Analogsinclude those which retain at least some of the biological activity ofthe wild-type polypeptide, e.g. blood clotting activity.

Polypeptide variants contemplated include, without limitation,polypeptides chemically modified by such techniques as ubiquitination,glycosylation, including polysialation, conjugation to therapeutic ordiagnostic agents, labeling, covalent polymer attachment such aspegylation (derivatization with polyethylene glycol), introduction ofnon-hydrolyzable bonds, and insertion or substitution by chemicalsynthesis of amino acids such as ornithine, which do not normally occurin human proteins. Variants retain the same or essentially the samebinding properties of non-modified molecules of the invention. Suchchemical modification may include direct or indirect (e.g., via, alinker) attachment of an agent to the VWF polypeptide. In the case ofindirect attachment, it is contemplated that the linker may behydrolyzable or non-hydrolyzable.

Preparing pegylated polypeptide analogs will in one aspect comprise thesteps of (a) reacting the polypeptide with polyethylene glycol (such asa reactive ester or aldehyde derivative of PEG) under conditions wherebythe binding construct polypeptide becomes attached to one or more PEGgroups, and (b) obtaining the reaction product(s). In general, theoptimal reaction conditions for the acylation reactions are determinedbased on known parameters and the desired result. For example, thelarger the ratio of PEG:protein, the greater the percentage ofpoly-pegylated product. In some embodiments, the binding construct has asingle PEG moiety at the N-terminus. Polyethylene glycol (PEG) may beattached to the blood clotting factor to, fir example, provide a longerhalf-life in Wyo. The PEG group may be of any convenient molecularweight and is linear or branched. The average molecular weight of thePEG ranges from about 2 kiloDalton (“kD”) to about 100 kDa, from about 5kDa to about 50 kDa, or from about 5 kDa to about 10 kDa. In certainaspects, the PEG groups are attached to the blood cloning factor viaacylation or reductive alkylation through a natural or engineeredreactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, orester group) to a reactive group on the blood clotting factor (e.g., analdehyde, amino, or ester group) or by any other technique known in theart.

Methods for preparing polysialylated polypeptide are described in UnitedStates Patent Publication 20060160948, Fernandes et Gregoriadis;Biochim. Biophys. Acta 1341: 26-34, 1997, and Saenko et al., Haemophilia12:42-51, 2006. Briefly, a solution of colominic acid (CA) containing0.1 M NaIO4 is stirred in the dark at room temperature to oxidize theCA. The activated CA solution is dialyzed against, e.g., 0.05 M sodiumphosphate buffer, pH 7.2 in the dark and this solution was added to arVWF solution and incubated for 18 h at room temperature in the darkunder gentle shaking. Free reagents are optionally be separated from therVWF-polysialic acid conjugate by, for example,ultrafiltration/diafiltration. Conjugation of rVWF with polysialic acidis achieved using glutaraldehyde as cross-linking reagent (Migneault etal., Biotechniques 37: 790-796, 2004).

It is further contemplated in another aspect that a polypeptide of theinvention is a fusion protein with a second agent which is apolypeptide. In one embodiment, the second agent which is a polypeptide,without limitation, is an enzyme, a growth factor, an antibody, acytokine, a chemokine, a cell-surface receptor, the extracellular domainof a cell surface receptor, a cell adhesion molecule, or fragment oractive domain of a protein described above. In a related embodiment, thesecond agent is a blood clotting factor such as Factor VIII, Factor VII,Factor IX. The fusion protein contemplated is made by chemical orrecombinant techniques well-known in the art.

It is also contemplated in another aspect that prepro-VWF and pro-VWFpolypeptides will provide a therapeutic benefit in the formulations ofthe present invention. For example, U.S. Pat. No. 7,005,502 describes apharmaceutical preparation comprising substantial amounts of pro-VWFthat induces thrombin gerneation in vitro. In addition to recombinant,biologically active fragments, variants, or other analogs of thenaturally-occurring mature VAT, the present invention contemplates theuse of recombinant biologically active fragments, variants, or analogsof the prepro-VWF (set out in SEQ NO:2) or pro-VWF polypeptides (aminoacid residues 23 to 764 of SEQ ID NO: 2) in the formulations describedherein.

Polynucleotides encoding fragments, variants and analogs may be readilygenerated by a worker of skill to encode biologically active fragments,variants, or analogs of the naturally-occurring molecule that possessthe same or similar biological activity to the naturally-occurringmolecule. In various aspects, these polynucleotides are prepared usingPCR techniques, digestion/ligation of DNA encoding molecule, and thelike. Thus, one of skill in the art will be able to generate single basechanges in the DNA strand to result in an altered codon and a missensemutation, using any method known in the art, including, but not limitedto site-specific mutagenesis. As used herein, the phrase “moderatelystringent hybridization conditions” means, for example, hybridization at42° C. in 50% formamide and washing at 60° C. in 0.1×SSC, 0.1% SDS. Itis understood by those of skill in the art that variation in theseconditions occurs based on the length and GC nucleotide base content ofthe sequences to be hybridized. Formulas standard in the art areappropriate for determining exact hybridization conditions. See Sambrooket al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

Lyophilization

In one aspect, the formulations comprising a VWF polypeptide of theinvention are lyophilized prior to administration. Lyophilization iscarried out using techniques common in the art and should be optimizedfor the composition being developed [Tang et al., Pharm Res. 21:191-200,(2004) and Chang et al., Pharm Res. 13:243-9 (1996)].

A lyophilization cycle is, in one aspect, composed of three steps:freezing, primary drying, and secondary drying [A. P. Mackenzie, PhilTrans R Soc London, Ser B, Biol 278:167 (1977)]. In the freezing step,the solution is cooled to initiate ice formation. Furthermore, this stepinduces the crystallization of the bulking agent. The ice sublimes inthe primary drying stage, which is conducted by reducing chamberpressure below the vapor pressure of the ice, using a vacuum andintroducing heat to promote sublimation. Finally, adsorbed or boundwater is removed at the secondary drying stage under reduced chamberpressure and at an elevated shelf temperature. The process produces amaterial known as a lyophilized cake. Thereafter the cake can bereconstituted with either sterile water or suitable diluent forinjection.

The lyophilization cycle not only determines the final physical state ofexcipients but also affects other parameters such as reconstitutiontime, appearance, stability and final moisture content. The compositionstructure in the frozen state proceeds through several transitions(e.g., glass transitions, wettings, and crystallizations) that occur atspecific temperatures and the structure may be used to understand andoptimize the lyophilization process. The glass transition temperature(Tg and/or Tg′) can provide information about the physical state of asolute and can be determined by differential scanning calorimetry (DSC).Tg and Tg′ are an important parameter that must be taken into accountwhen designing the lyophilization cycle. For example, Tg′ is importantfor primary drying. Furthermore, in the dried state, the glasstransition temperature provides information on the storage temperatureof the final product.

Formulations and Excipients in General

Excipients are additives that either impart or enhance the stability anddelivery of a drug product (e.g., protein). Regardless of the reason fortheir inclusion, excipients are an integral component of a formulationand therefore need to be safe and well tolerated by patients. Forprotein drugs, the choice of excipients is particularly importantbecause they can affect both efficacy and immunogenicity of the drug.Hence, protein formulations need to be developed with appropriateselection of excipients that afford suitable stability, safety, andmarketability.

A lyophilized formulation is, in one aspect, at least comprised of oneor more of a buffer, a bulking agent, and a stabilizer. In this aspect,the utility of a surfactant is evaluated and selected in cases whereaggregation during the lyophilization step or during reconstitutionbecomes an issue. An appropriate buffering agent is included to maintainthe formulation within stable zones of pH during lyophilization. Acomparison of the excipient components contemplated for liquid andlyophilized protein formulations is provided in Table A.

TABLE A Excipient components of lyophilized protein formulationsExcipient component Function in lyophilized formulation Buffer MaintainpH of formulation during lyophilization and upon reconstitution Tonicityagent/ Stabilizers include cryo and lyoprotectants stabilizer Examplesinclude Polyols, sugars and polymers Cryoprotectants protect proteinsfrom freezing stresses Lyoprotectants stabilize proteins in thefreeze-dried state Bulking agent Used to enhance product elegance and toprevent blowout Provides structural strength to the lyo cake Examplesinclude mannitol and glycine Surfactant Employed if aggregation duringthe lyophilization process is an issue May serve to reducereconstitution times Examples include polysorbate 20 and 80 Anti-oxidantUsually not employed, molecular reactions in the lyo cake are greatlyretarded Metal ions/ May be included if a specific metal ion is includedonly chelating agent as a co-factor or where the metal is required forprotease activity Chelating agents are generally not needed in lyoformu- lations Preservative For multi-dose formulations only Providesprotection against microbial growth in formu- lation Is usually includedin the reconstitution diluent (e.g. bWFI)

The principal challenge in developing formulations for proteins isstabilizing the product against the stresses of manufacturing, shippingand storage. The role of formulation excipients is to providestabilization against these stresses. Excipients are also be employed toreduce viscosity of high concentration protein formulations in order toenable their delivery and enhance patient convenience. In general,excipients can be classified on the basis of the mechanisms by whichthey stabilize proteins against various chemical and physical stresses.Some excipients are used to alleviate the effects of a specific stressor to regulate a particular susceptibility of a specific protein. Otherexcipients have more general effects on the physical and covalentstabilities of proteins. The excipients described herein are organizedeither by their chemical type or their functional role in formulations.Brief descriptions of the modes of stabilization are provided whendiscussing each excipient type.

Given the teachings and guidance provided herein, those skilled in theart will know what amount or range of excipient can be included in anyparticular formulation to achieve a biopharmaceutical formulation of theinvention that promotes retention in stability of the biopharmaceutical(e.g., a protein). For example, the amount and type of a salt to beincluded in a biopharmaceutical formulation of the invention is selectedbased on the desired osmolality (i.e., isotonic, hypotonic orhypertonic) of the final solution as well as the amounts and osmolalityof other components to be included in the formulation.

By way of example, inclusion of about 5% sorbitol can achieveisotonicity while about 9% of a sucrose excipient is needed to achieveisotonicity. Selection of the amount or range of concentrations of oneor more excipients that can be included within a biopharmaceuticalformulation of the invention has been exemplified above by reference tosalts, polyols and sugars. However, those skilled in the art willunderstand that the considerations described herein and furtherexemplified by reference to specific excipients are equally applicableto all types and combinations of excipients including, for example,salts, amino acids, other tonicity agents, surfactants, stabilizers,bulking agents, cryoprotectants, lyoprotectants, anti-oxidants, metalions, chelating agents and/or preservatives.

Further, where a particular excipient is reported in molarconcentration, those skilled in the art will recognize that theequivalent percent (%) w/v (e.g., (grams of substance in a solutionsample/mL of solution)×100%) of solution is also contemplated.

Of course, a person having ordinary skill in the art would recognizethat the concentrations of the excipients described herein share aninterdependency within a particular formulation. By way of example, theconcentration of a bulking agent may be lowered where, e.g., there is ahigh protein concentration or where, e.g., there is a high stabilizingagent concentration. In addition, a person having ordinary skill in theart would recognize that, in order to maintain the isotonicity of aparticular formulation in which there is no bulking agent, theconcentration of a stabilizing agent would be adjusted accordingly(i.e., a “tonicifying” amount of stabilizer would be used). Commonexcipients are known in the art and can be found in Powell et al.,Compendium of Excipients fir Parenteral Formulations (1998), PDA J.Pharm. Sci. Technology, 52:238-311.

Buffers and Buffering Agents

The stability of a pharmacologically active protein formulation isusually observed to be maximal in a narrow pH range. This pH range ofoptimal stability needs to be identified early during pre-formulationstudies. Several approaches, such as accelerated stability studies andcalorimetric screening studies, are useful in this endeavor (Remmele R.L. Jr., et al., Biochemistry, 38(16): 5241-7 (1999)). Once a formulationis finalized, the protein must be manufactured and maintained throughoutits shelf-life. Hence, buffering agents are almost always employed tocontrol pH in the formulation.

The buffer capacity of the buffering species is maximal at a pH equal tothe pKa and decreases as pH increases or decreases away from this value.Ninety percent of the buffering capacity exists within one pH unit ofits pKa. Buffer capacity also increases proportionally with increasingbuffer concentration.

Several factors need to be considered when choosing a buffer. First andforemost, the buffer species and its concentration need to be definedbased on its pKa and the desired formulation pH. Equally important is toensure that the buffer is compatible with the protein and otherformulation excipients, and does not catalyze any degradation reactions.A third important aspect to be considered is the sensation of stingingand irritation the buffer may induce upon administration. For example,citrate is known to cause stinging upon injection (Laursen T, et al.,Basic Clin Pharmacol Toxicol., 98(2): 218-21 (2006)). The potential forstinging and irritation is greater for drugs that are administered viathe subcutaneous (SC) or intramuscular (IM) routes, where the drugsolution remains at the site for a relatively longer period of time thanwhen administered by the IV route where the formulation gets dilutedrapidly into the blood upon administration. For formulations that areadministered by direct IV infusion, the total amount of buffer (and anyother formulation component) needs to be monitored. One has to beparticularly careful about potassium ions administered in the form ofthe potassium phosphate buffer, which can induce cardiovascular effectsin a patient (Hollander-Rodriguez J C, et al., Am. Fam. Physician.,73(2): 283-90 (2006)).

Buffers for lyophilized formulations need additional consideration. Somebuffers like sodium phosphate can crystallize out of the proteinamorphous phase during freezing resulting in shifts in pH. Other commonbuffers such as acetate and imidazole may sublime or evaporate duringthe lyophilization process, thereby shifting the pH of formulationduring lyophilization or after reconstitution.

The buffer system present in the compositions is selected to bephysiologically compatible and to maintain a desired pH of thepharmaceutical formulation. In one embodiment, the pH of the solution isbetween pH 2.0 and pH 12.0. For example, the pH of the solution may be2.0, 2.3, 2.5, 2.7, 3.0, 3.3, 3.5, 3.7, 4.0, 4.3, 4.5, 4.7, 5.0, 5.3,5.5, 5.7, 6.0, 6.3, 6.5, 6.7, 7.0, 7.3, 7.5, 7.7, 8.0, 8.3, 8.5, 8.7,9.0, 9.3, 9.5, 9.7, 10.0, 10.3, 10.5, 10.7, 11.0, 11.3, 11.5, 11.7, or12.0.

The pH buffering compound may be present in any amount suitable tomaintain the pH of the formulation at a predetermined level. In oneembodiment, the pH buffering concentration is between 0.1 mM and 500 mM(1 M). For example, it is contemplated that the pH buffering agent is atleast 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70,80, 90, 100, 200, or 500 mM.

Exemplary pH buffering agents used to buffer the formulation as set outherein include, but are not limited to organic acids, glycine,histidine, glutamate, succinate, phosphate, acetate, citrate, Tris,HEPES, and amino acids or mixtures of amino acids, including, but notlimited to aspartate, histidine, and glycine. In one embodiment of thepresent invention, the buffering agent is citrate.

Stabilizers and Bulking Agents

In one aspect of the present pharmaceutical formulations, a stabilizer(or a combination of stabilizers) is added to prevent or reducestorage-induced aggregation and chemical degradation. A hazy or turbidsolution upon reconstitution indicates that the protein has precipitatedor at least aggregated. The term “stabilizer” means an excipient capableof preventing aggregation or physical degradation, including chemicaldegradation (for example, autolysis, deamidation, oxidation, etc.) in anaqueous state. Stabilizers contemplated include, but are not limited to,sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose,cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose,mannitol, sorbitol, glycine, arginine HCL, poly-hydroxy compounds,including polysaccharides such as dextran, starch, hydroxyethyl starch,cyclodextrins, N-methyl pyrollidene, cellulose and hyaluronic acid,sodium chloride, [Carpenter et al., Develop. Biol. Standard 74:225,(1991)]. In the present formulations, the stabilizer is incorporated ina concentration of about 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 17, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM. In oneembodiment f the present invention, mannitol and trehalose are used asstabilizing agents.

If desired, the formulations also include appropriate amounts of bulkingand osmolarity regulating agents. Bulking agents include, for exampleand without limitation, mannitol, glycine, sucrose, polymers such asdextran, polyvinylpyrolidone, carboxymethylcellulose, lactose, sorbitol,trehalose, or xylitol. In one embodiment, the bulking agent is mannitol.The bulking agent is incorporated in a concentration of about 0.1, 0.5,0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,500, 700, 900, or 1000 mM.

Surfactants

Proteins have a high propensity to interact with surfaces making themsusceptible to adsorption and denaturation at air-liquid, vial-liquid,and liquid-liquid (silicone oil) interfaces. This degradation pathwayhas been observed to be inversely dependent on protein concentration andresults in either the formation of soluble and insoluble proteinaggregates or the loss of protein from solution via adsorption tosurfaces. In addition to container surface adsorption, surface-induceddegradation is exacerbated with physical agitation, as would beexperienced during shipping and handling of the product.

Surfactants are commonly used in protein formulations to preventsurface-induced degradation. Surfactants are amphipathic molecules withthe capability of out-competing proteins for interfacial positions.Hydrophobic portions of the surfactant molecules occupy interfacialpositions (e.g., air/liquid), while hydrophilic portions of themolecules remain oriented towards the bulk solvent. At sufficientconcentrations (typically around the detergent's ethical micellarconcentration), a surface layer of surfactant molecules serve to preventprotein molecules from adsorbing at the interface. Thereby,surface-induced degradation is minimized. Surfactants contemplatedherein include, without limitation, fatty acid esters of sorbitanpolyethoxylates, i.e. polysorbate 20 and polysorbate 80. The two differonly in the length of the aliphatic chain that imparts hydrophobiccharacter to the molecules, C-12 and C-18, respectively. Accordingly,polysorbate-80 is more surface-active and has a lower critical micellarconcentration than polysorbate-20.

Detergents can also affect the thermodynamic conformational stability ofproteins. Here again, the effects of a given detergent excipient will beprotein specific. For example, polysorbates have been shown to reducethe stability of some proteins and increase the stability of others.Detergent destabilization of proteins can be rationalized in terms ofthe hydrophobic tails of the detergent molecules that can engage inspecific binding with partially or wholly unfolded protein states. Thesetypes of interactions could cause a shift in the conformationalequilibrium towards the more expanded protein states (i.e. increasingthe exposure of hydrophobic portions of the protein molecule incomplement to binding polysorbate). Alternatively, if the protein nativestate exhibits some hydrophobic surfaces, detergent binding to thenative state may stabilize that conformation.

Another aspect of polysorbates is that they are inherently susceptibleto oxidative degradation. Often, as raw materials, they containsufficient quantities of peroxides to cause oxidation of protein residueside-chains, especially methionine. The potential for oxidative damagearising from the addition of stabilizer emphasizes the point that thelowest effective concentrations of excipients should be used informulations. For surfactants, the effective concentration for a givenprotein will depend on the mechanism of stabilization.

Surfactants are also added in appropriate amounts to prevent surfacerelated aggregation phenomenon during freezing and drying [Chang, B, J.Pharm. Sci. 85:1325, (1996)]. Thus, exemplary surfactants include,without limitation, anionic, cationic, nonionic, zwitterionic, andamphoteric surfactants including surfactants derived fromnaturally-occurring amino acids. Anionic surfactants include, but arenot limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate anddioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosinesodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt,sodium cholate hydrate, sodium deoxycholate, and glycodeoxycholic acidsodium salt. Cationic surfactants include, but are not limited to,benzalkonium chloride or benzethonium chloride, cetylpyridinium chloridemonohydrate, and hexadecyltrimethylammonium bromide. Zwitterionicsurfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, andSB3-12. Non-ionic surfactants include, but are not limited to,digitonin, Triton X-100, Triton X-114, TWEEN-20, and TWEEN-80.Surfactants also include, but are not limited to lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10.40, 50and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, soylecithin and other phospholipids such as dioleyl phosphatidyl choline(DOPC), dimyristoylphosphatidyl glycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), and (dioleyl phosphatidyl glycerol) DOPG; sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Compositionscomprising these surfactants, either individually or as a mixture indifferent ratios, are therefore further provided. In one embodiment ofthe present invention, the surfactant is TWEEN-80. In the presentformulations, the surfactant is incorporated in a concentration of about0.01 to about 0.5 g/L. In formulations provided, the surfactantconcentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 g/L.

Salts

Salts are often added to increase the ionic strength of the formulation,which can be important for protein solubility, physical stability, andisotonicity. Salts can affect the physical stability of proteins in avariety of ways. Ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface. Alternatively,salts can stabilize the denatured state by binding to peptide groupsalong the protein backbone (—CONH—). Salts can also stabilize theprotein native conformation by shielding repulsive electrostaticinteractions between residues within a protein molecule. Salts inprotein formulations can also shield attractive electrostaticinteractions between protein molecules that can lead to proteinaggregation and insolubility. In formulations provided, the saltconcentration is between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150,200, 300, and 500 mM.

Other Common Excipient Components

Amino Acids

Amino acids have found versatile use in protein formulations as buffers,bulking agents, stabilizers and antioxidants. Thus, in one aspecthistidine and glutamic acid are employed to buffer protein formulationsin the pH range of 5.5-6.5 and 4.0-5.5 respectively. The imidazole groupof histidine has a pKa=6.0 and the carboxyl group of glutamic acid sidechain has a pKa of 4.3 which makes these amino acids suitable forbuffering in their respective pH ranges. Glutamic acid is particularlyuseful in such cases. Histidine is commonly found in marketed proteinformulations, and this amino acid provides an alternative to citrate, abuffer known to sting upon injection. Interestingly, histidine has alsobeen reported to have a stabilizing effect, with respect to aggregationwhen used at high concentrations in both liquid and lyophilizedpresentations (Chen B, et al., Pharm Res., 20(12): 1952-60 (2003)),Histidine was also observed by others to reduce the viscosity of a highprotein concentration formulation. However, in the same study, theauthors observed increased aggregation and discoloration in histidinecontaining formulations during freeze-thaw studies of the antibody instainless steel containers. Another note of caution with histidine isthat it undergoes photo-oxidation in the presence of metal ions (TomitaM, et al., Biochemistry, 8(12): 5149-60 (1969)). The use of methionineas an antioxidant in formulations appears promising; it has beenobserved to be effective against a number of oxidative stresses (Lam XM, et al., J Pharm Set., 86(11): 1250-5 (1997)).

In various aspects, formulations are provided which include one or moreof the amino acids glycine, proline, serine, arginine and alanine havebeen shown to stabilize proteins by the mechanism of preferentialexclusion. Glycine is also a commonly used bulking agent in lyophilizedformulations. Arginine has been shown to be an effective agent ininhibiting aggregation and has been used in both liquid and lyophilizedformulations.

In formulations provided, the amino acid concentration is between 0.1,1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300, and 500 mM. In oneembodiment of the present invention, the amino acid is glycine.

Antioxidants

Oxidation of protein residues arises from a number of different sources.Beyond the addition of specific antioxidants, the prevention ofoxidative protein damage involves the careful control of a number offactors throughout the manufacturing process and storage of the productsuch as atmospheric oxygen, temperature, light exposure, and chemicalcontamination. The invention therefore contemplates the use of thepharmaceutical antioxidants including, without limitation, reducingagents, oxygen/free-radical scavengers, or chelating agents.Antioxidants in therapeutic protein formulations are; in one aspect,water-soluble and remain active throughout the product shelf-life.Reducing agents and oxygen/free-radical scavengers work by ablatingactive oxygen species in solution. Chelating agents such as EDTA areeffective by binding trace metal contaminants that promote free-radicalformation. For example, EDTA was utilized in the liquid formulation ofacidic fibroblast growth factor to inhibit the metal ion catalyzedoxidation of cysteine residues.

In addition to the effectiveness of various excipients to preventprotein oxidation, the potential for the antioxidants themselves toinduce other covalent or physical changes to the protein is of concern.For example, reducing agents can cause disruption of intramoleculardisulfide linkages, which can lead to disulfide shuffling, in thepresence of transition metal ions, ascorbic acid and EDTA have beenshown to promote methionine oxidation in a number of proteins andpeptides (Akers M J, and Defelippis M R. Peptides and Proteins asParenteral Solutions. In: Pharmaceutical Formulation Development ofPeptides and Proteins. Sven Frokjaer, Lars Hovgaard, editors.Pharmaceutical Science. Taylor and Francis, UK (1999)), Fransson J. R.,J. Pharm. Sci. 86(9): 4046-1050 (1997), Yin J, et al., Pharm Res.,21(12): 2377-83 (2004)). Sodium thiosulfate has been reported to reducethe levels of light and temperature induced methionine-oxidation inrhuMab HER2; however, the formation of a thiosulfate-protein adduct wasalso reported in this study (Lam X M, Yang J Y, et al., J Pharm Sci.86(11): 1250-5 (1997)). Selection of an appropriate antioxidant is madeaccording to the specific stresses and sensitivities of the protein.Antioxidants contemplated in certain aspects include, withoutlimitation, reducing agents and oxygen/free-radical scavengers, EDTA,and sodium thiosulfate.

Metal Ions

In general, transition metal ions are undesired in protein formulationsbecause they can catalyze physical and chemical degradation reactions inproteins. However, specific metal ions are included in formulations whenthey are co-factors to proteins and in suspension formulations ofproteins where they form coordination complexes (e.g., zinc suspensionof insulin). Recently, the use of magnesium ions (10-120 mM) has beenproposed to inhibit the isomerization of aspartic acid to isoasparticacid (WO 2004039337).

Two examples where metal ions confer stability or increased activity inproteins are human deoxyribonuclease (rhDNase, Pulmozyme®), and FactorVIII. In the case of rhDNase, Ca⁺² ions (up to 100 mM) increased thestability of the enzyme through a specific binding site (Chen B, et al.,J Pharm Sci., 88(4): 477-82 (1999)). In fact, removal of calcium ionsfrom the solution with EGTA caused an increase in deamidation andaggregation. However, this effect was observed only with Ca⁺² ions:other divalent cations Mg⁺², Mn⁺² and Zn⁺² were observed to destabilizerhDNase. Similar effects were observed in Factor VIII, Ca⁺² and Sr⁺²ions stabilized the protein while others like Mg⁺², Mn⁺² and Zn⁺², Cu⁺²and Fe⁺² destabilized the enzyme (Fatouros, A., et at, Int. J. Pharm.,155, 1.21-131 (1997). In a separate study with Factor VIII, asignificant increase in aggregation rate was observed in the presence ofAl⁺³ ions (Derrick T S, et al., J. Pharm. Sci., 93(10): 2549-57 (2004)).The authors note that other excipients like buffer salts are oftencontaminated with Al⁺³ ions and illustrate the need to use excipients ofappropriate quality in formulated products.

Preservatives

Preservatives are necessary when developing multi-use parenteralformulations that involve more than one extraction from the samecontainer. Their primary function is to inhibit microbial growth andensure product sterility throughout the shelf-life or term of use of thedrug product. Commonly used preservatives include, without limitation,benzyl alcohol, phenol and m-cresol. Although preservatives have a longhistory of use, the development of protein formulations that includespreservatives can be challenging. Preservatives almost always have adestabilizing effect (aggregation) on proteins, and this has become amajor factor in limiting their use in multi-dose protein formulations(Roy S, et al., J Pharm Sci., 94(2): 382-96 (2005)).

To date, most protein drugs have been formulated for single-use only.However, when multi-dose formulations are possible, they have the addedadvantage of enabling patient convenience, and increased marketability.A good example is that of human growth hormone (hGH) where thedevelopment of preserved formulations has led to commercialization ofmore convenient, multi-use injection pen presentations. At least foursuch pen devices containing preserved formulations of hGH are currentlyavailable on the market. Norditropin® (liquid, Novo Nordisk), NutropinAQ® (liquid, Genentech) &. Genotropin (lyophilized—dual chambercartridge, Pharmacia & Upjohn) contain phenol while Somatrope® (EliLilly) is formulated with in-cresol.

Several aspects need to be considered during the formulation developmentof preserved dosage forms. The effective preservative concentration inthe drug product must be optimized. This requires testing a givenpreservative in the dosage form with concentration ranges that conferanti-microbial effectiveness without compromising protein stability. Forexample, three preservatives were successfully screened in thedevelopment of a liquid formulation for interleukin-1 receptor (Type 1),using differential scanning calorimetry (DSC). The preservatives wererank ordered based on their impact on stability at concentrationscommonly used in marketed products (Remmele R L Jr., et al., Pharm Res.,15(2): 200-8 (1998)).

Development of liquid formulations containing preservatives are morechallenging than lyophilized formulations. Freeze-dried products can belyophilized without the preservative and reconstituted with apreservative containing diluent at the time of use. This shortens thetime for which a preservative is in contact with the proteinsignificantly minimizing the associated stability risks. With liquidformulations, preservative effectiveness and stability have to bemaintained over the entire product shelf-life (˜18-24 months). Animportant point to note is that preservative effectiveness has to bedemonstrated in the final formulation containing the active drug and allexcipient components.

Some preservatives can cause injection site reactions, which is anotherfactor that needs consideration when choosing a preservative. Inclinical trials that focused on the evaluation of preservatives andbuffers in Norditropin, pain perception was observed to be lower informulations containing phenol and benzyl alcohol as compared to aformulation containing m-cresol (Kappelgaard A. M., Horny Res. 62 Suppl3:98-103 (2004)). Interestingly, among the commonly used preservative,benzyl alcohol possesses anesthetic properties (Minogue S C, and Sun DA., Anesth Analg., 100(3): 683-6 (2005)). In various aspects the use ofpreservatives provide a benefit that outweighs any side effects.

Methods of Preparation

The present invention further contemplates methods for the preparationof pharmaceutical formulations.

The present methods further comprise one or more of the following steps:adding a stabilizing agent as described herein to said mixture prior tolyophilizing, adding at least one agent selected from a bulking agent,an osmolarity regulating agent, and a surfactant, each of which asdescribed herein, to said mixture prior to lyophilization.

The standard reconstitution practice for lyophilized material is to addback a volume of pure water or sterile water for injection (WFI)(typically equivalent to the volume removed during lyophilization),although dilute solutions of antibacterial agents are sometimes used inthe production of pharmaceuticals for parenteral administration [Chen,Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)].Accordingly, methods are provided for preparation of reconstituted rVWFcompositions comprising the step of adding a diluent to a lyophilizedrVWF composition of the invention.

The lyophilized material may be reconstituted as an aqueous solution, Avariety of aqueous carriers, e.g., sterile water for injection, waterwith preservatives for multi dose use, or water with appropriate amountsof surfactants (for example, an aqueous suspension that contains theactive compound in admixture with excipients suitable for themanufacture of aqueous suspensions). In various aspects, such excipientsare suspending agents, for example and without limitation, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents are a naturally-occurring phosphatide, forexample and without limitation, lecithin, or condensation products of analkylene oxide with fatty acids, for example and without limitation,polyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohols, for example and without limitation,heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatly acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatly acids and hexitolanhydrides, for example and without limitation, polyethylene sorbitanmonooleate. In various aspects, the aqueous suspensions also contain oneor more preservatives, for example and without limitation, ethyl, orn-propyl, p-hydroxybenzoate.

Administration

To administer compositions to human or test animals, in one aspect, thecompositions comprises one or more pharmaceutically acceptable carriers.The phrases “pharmaceutically” or “pharmacologically” acceptable referto molecular entities and compositions that are stable, inhibit proteindegradation such as aggregation and cleavage products, and in additiondo not produce allergic, or other adverse reactions when administeredusing routes well-known in the art, as described below.“Pharmaceutically acceptable carriers” include any and all clinicallyuseful solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like,including those agents disclosed above.

The pharmaceutical formulations are administered orally, topically,transdermally, parenterally, by inhalation spray, vaginally, rectally,or by intracranial injection. The term parenteral as used hereinincludes subcutaneous injections, intravenous, intramuscular,intracisternal injection, or infusion techniques. Administration byintravenous, intradermal, intramuscular, intramammary, intraperitoneal,intrathecal, retrobulbar, intrapulmonary injection and or surgicalimplantation at a particular site is contemplated as well. Generally,compositions are essentially free of pyrogens, as well as otherimpurities that could be harmful to the recipient.

Single or multiple administrations of the compositions are carried outwith the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage depends on the type of disease to be treated, as defined above,the severity and course of the disease, whether drug is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the drug, and the discretion of theattending physician.

Kits

As an additional aspect, the invention includes kits which comprise oneor more lyophilized compositions packaged in a manner which facilitatestheir use for administration to subjects. In one embodiment, such a kitincludes pharmaceutical formulation described herein (e.g., acomposition comprising a therapeutic protein or peptide), packaged in acontainer such as a sealed bottle or vessel, with a label affixed to thecontainer or included in the package that describes use of the compoundor composition in practicing the method. In one embodiment, thepharmaceutical formulation is packaged in the container such that theamount of headspace in the container (e.g., the amount of air betweenthe liquid formulation and the top of the container) is very small.Preferably, the amount of headspace is negligible (i.e., almost none).In one embodiment, the kit contains a first container having atherapeutic protein or peptide composition and a second container havinga physiologically acceptable reconstitution solution for thecomposition. In one aspect, the pharmaceutical formulation is packagedin a unit dosage form. The kit may further include a device suitable foradministering the pharmaceutical formulation according to a specificroute of administration. Preferably, the kit contains a label thatdescribes use of the pharmaceutical formulations.

Dosages

The dosage regimen involved in a method for treating a conditiondescribed herein will be determined by the attending physician,considering various factors which modify the action of drugs, e.g. theage, condition, body weight, sex and diet of the patient, the severityof any infection, time of administration and other clinical factors. Byway of example, a typical dose of a recombinant VWF of the presentinvention is approximately 50 U/kg, equal to 500 μg/kg.

In one aspect, formulations of the invention are administered by aninitial bolus followed by a continuous infusion to maintain therapeuticcirculating levels of drug product. As another example, the inventivecompound is administered as a one-time dose. Those of ordinary skill inthe art will readily optimize effective dosages and administrationregimens as determined by good medical practice and the clinicalcondition of the individual patient. The frequency of dosing depends onthe pharmacokinetic parameters of the agents and the route ofadministration. The optimal pharmaceutical formulation is determined byone skilled in the art depending upon the route of administration anddesired dosage. See for example, Remington's Pharmaceutical Sciences,18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712,the disclosure of which is hereby incorporated by reference. Suchformulations influence the physical state, stability, rate of in vivorelease, and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose is calculatedaccording to body weight, body surface area or organ size. Appropriatedosages may be ascertained through use of established assays fordetermining blood level dosages in conjunction with appropriatedose-response data. The final dosage regimen is determined by theattending physician, considering various factors which modify the actionof drugs, e.g. the drug's specific activity, the severity of the damageand the responsiveness of the patient, the age, condition, body weight,sex and diet of the patient, the severity of any infection, time ofadministration and other clinical factors. As studies are conducted,further information will emerge regarding the appropriate dosage levelsand duration of treatment for various diseases and conditions.

The following examples are not intended to be limiting but onlyexemplary of specific embodiments of the invention.

EXAMPLE 1 Shaking Experiments

In order to determine the amount of precipitation of rVWF in variousformulations, the extent of aggregation of rVWF following turbulentshaking was tested under a variety of conditions.

As shown in Table 1 below, various rVWF formulations were assessed in a20 mM citrate buffer, pH 7.3. Shaking experiments were designed tosimulate mechanical stress conditions. 1-2 ml of each formulation wasshaken with a laboratory shaker for 10 minutes at 1200 rpm.

TABLE 1 PEG Tween Lyo 25 Lysine Histidine Glycine Serine Mannitol 150080 Sucrose Trehalose Raffinose 18 30 mM 5 g/L 19 30 mM 5 g/L 20 30 mM 5g/L 21 30 mM 5 g/L 22 30 mM 5 g/L 23 30 mM 5 g/L 24 30 mM 5 g/L 25 30 mM5 g/L 26 30 mM 0.1 g/L 27 30 mM 0.1 g/L 28 30 mM 0.1 g/L 29 30 mM 0.1g/L 30 30 mM 5 g/L 5 g/L 31 30 mM 5 g/L 5 g/L 32 30 mM 5 g/L 5 g/L 33 30mM 5 g/L 5 g/L 34 30 mM 5 g/L 0.1 g/L 35 30 mM 5 g/L 0.1 g/L 36 30 mM 5g/L 0.1 g/L 37 30 mM 5 g/L 0.1 g/L 38 30 mM 5 g/L 5 g/L 0.1 g/L 39 30 mM5 g/L 5 g/L 0.1 g/L 40 30 mM 5 g/L 5 g/L 0.1 g/L 41 30 mM 5 g/L 5 g/L0.1 g/L 42 5 g/L 43 5 g/L 44 5 g/L 45 5 g/L 46 5 g/L 5 g/L 47 5 g/L 5g/L 48 5 g/L 5 g/L 49 5 g/L 5 g/L 50 5 g/L 0.1 g/L 51 0.1 g/L 5 g/L 520.1 g/L 5 g/L 53 0.1 g/L 5 g/L

The assessment of the visible VWF aggregates was done according to thescheme shown below. “Visible aggregates,” in most cases, are gelatinousfibers ranging in size from about 100 nm to 1-2 cm.

SCHEME Particles A no particles B several particles, rarely visible(dots) B1 many particles, rarely visible (dots) C several particles,easily visible (fibers) D many particles, easily visible (fibers) Evisible particles (>1 mm fibers) E1 fluffy white precipitate (swims onthe surface) E2 jellyfish

The results of the shaking experiments are shown in Table 2, below.

TABLE 2 Shaking 1-2 mL Lyo 25 PEG Tween 1200 rpm Samples LysineHistidine Glycine Serine Mannitol 1500 80 30 min 18 30 mM 5 g/L E1 19 30mM 5 g/L E1 20 30 mM 5 g/L E1 21 30 mM 5 g/L E1 22 30 mM 5 g/L E1 23 30mM 5 g/L E1 24 30 mM 5 g/L E1 25 30 mM 5 g/L E1 26 30 mM 0.1 g/L E2 big27 30 mM 0.1 g/L E2 big 28 30 mM 0.1 g/L E2 ~6 mm 29 30 mM 0.1 g/L E2 ~3mm 30 30 mM 5 g/L 5 g/L E1 31 30 mM 5 g/L 5 g/L E1 32 30 mM 5 g/L 5 g/LE1 33 30 mM 5 g/L 5 g/L E1 34 30 mM 5 g/L 0.1 g/L B1 35 30 mM 5 g/L 0.1g/L B 36 30 mM 5 g/L 0.1 g/L E2 big 37 30 mM 5 g/L 0.1 g/L E2 big 38 30mM 5 g/L 5 g/L 0.1 g/L D 39 30 mM 5 g/L 5 g/L 0.1 g/L D 40 30 mM 5 g/L 5g/L 0.1 g/L B 41 30 mM 5 g/L 5 g/L 0.1 g/L B

In summary, the shaking experiments described above indicate thatformulations containing TWEEN-80® and Mannitol provide the best results(i.e., the least amount of aggregation).

EXAMPLE 2 Freeze-Thaw Experiments

Freeze-thaw experiments were designed to assess the impact of stresscaused by repeated freezing and thawing. In addition to the formulationsdescribed above for the shaking experiments (Table 1), the followingformulations were assessed (Table 3 and Table 4):

TABLE 3 Lyo 25 PEG Tween Samples Mannitol 1500 80 Sucrose TrehaloseRaffinose 42 5 g/L 43 5 g/L 44 5 g/L 45 5 g/L 46 5 g/L 5 g/L 47 5 g/L 485 g/L 49 5 g/L 50 5 g/L 0.1 g/L 51 0.1 g/L 5 g/L 52 0.1 g/L 5 g/L 53 0.1g/L 5 g/L

TABLE 4 Lyo 25 PEG Samples Lysine Histidine Glycine Serine Mannitol 1500Tween-80 Sucrose Trehalose Raffinose 76 20 g/L 0.2 g/L 20 g/L 77 20 g/L0.2 g/L 10 g/L 78 20 g/L 0.2 g/L 10 g/L 79 15 mM 20 g/L 0.2 g/L 20 g/L80 15 mM 20 g/L 0.2 g/L 10 g/L 81 15 mM 20 g/L 0.2 g/L 10 g/L 82 15 mM15 mM 20 g/L 0.2 g/L 20 g/L 83 15 mM 15 mM 20 g/L 0.2 g/L 10 g/L 84 15mM 15 mM 20 g/L 0.2 g/L 10 g/L 85 15 mM 15 mM 20 g/L 5 g/L 0.2 g/L 86 15mM 15 mM 20 g/L 5 g/L 0.2 g/L 10 g/L 87 15 mM 15 mM 20 g/L 15 g/L  0.2g/L 10 g/L 88 15 mM 15 mM 20 g/L 0.2 g/L  5 g/L 89 15 mM 15 mM 20 g/L0.2 g/L 15 g/L 90 15 mM 20 g/L 0.2 g/L 15 g/L 92 15 mM 15 mM 20 g/L 0.2g/L 10 g/L 93 30 mM 20 g/L 0.2 g/L 10 g/L 94 30 mM 20 g/L 0.2 g/L 10 g/L95 30 mM 20 g/L 0.2 g/L 10 g/L 96 30 mM 20 g/L 0.2 g/L 10 g/L 97 15 mM20 g/L 0.2 g/L 10 g/L 98 15 mM 15 mM 20 g/L 0.2 g/L 10 g/L 99 15 mM 15mM 20 g/L 0.2 g/L  5 g/L 100 15 mM 20 g/L 0.2 g/L 10 g/L 15 mM 20 g/L0.2 g/L 10 g/L

All formulations were frozen at −20° C. in a freezer for approximately 1hour and then thawed at room temperature. The results are shown in Table5 below.

TABLE 5 Freeze/ Freeze/ Thaw Lyo 25 PEG Tween- Thaw (~10 Samples LysineHistidine Glycine Serine Mannitol 1500 80 Sucrose Trehalose Raffinose (4times) times) 18 30 mM 5 g/L E1 15 19 30 mM 5 g/L E1 19 20 30 mM 5 g/L C20 21 30 mM 5 g/L C 21 22 30 mM 5 g/L C 22 23 30 mM 5 g/L C/B1 23 24 30mM 5 g/L C 24 25 30 mM 5 g/L C 25 26 30 mM 0.1 g/L B 26 27 30 mM 0.1 g/LB-B1 27 28 30 mM 0.1 g/L B 28 29 30 mM 0.1 g/L E 29 30 30 mM 5 g/L 5 g/LE 30 31 30 mM 5 g/L 5 g/L D 31 32 30 mM 5 g/L 5 g/L B1-C 32 33 30 mM 5g/L 5 g/L C/D 33 34 30 mM 5 g/L 0.1 g/L E2 (rest B) 34 35 30 mM 5 g/L0.1 g/L E 35 36 30 mM 5 g/L 0.1 g/L E 36 37 30 mM 5 g/L 0.1 g/L B 37 3830 mM 5 g/L 5 g/L 0.1 g/L B 38 39 30 mM 5 g/L 5 g/L 0.1 g/L B 39 40 30mM 5 g/L 5 g/L 0.1 g/L B 40 41 30 mM 5 g/L 5 g/L 0.1 g/L A 41 42 5 g/L D42 43 5 g/L D 43 44 5 g/L E1 44 45 5 g/L E1 45 46 5 g/L 5 g/L D 46 47 5g/L 5 g/L D 47 48 5 g/L 5 g/L D-E 48 49 5 g/L 5 g/L E 49 50 5 g/L 0.1g/L B1 50 51 0.1 g/L 5 g/L C-D 51 52 0.1 g/L 5 g/L B1 52 53 0.1 g/L 5g/L B1 53

As shown above, Trehalose provided the best results (i.e., the leastamount of aggregation).

EXAMPLE 3 Lyophilization Experiments

Lyophilization experiments were designed to assess the ability 0 variousformulations to allow the formation of a lyo-cake which dissolves inless than 10 minutes and results in a clear solution. An acceleratedstability study was also performed to demonstrate that no significantloss of biological activity.

The formulations shown in Table 6 below were lyophilized with a nitrogenlyophilizer TS20002 according to the manufacturer's instructions. Thetotal time for lyophilization was approximately 72 hours, Each of theformulations below also contained 20 g/L Mannitol and 0.1 g/L TWEEN-80®.

TABLE 6 Lyo 26 Citrate HEPES Glycine Histidine Acetate Tris PhosphateLysine Histidine Glycine Trehalose Raffinose 1 15 mM 10 g/L 2 15 mM 15mM 10 g/L 3 15 mM 15 mM 10 g/L 4 15 mM 15 mM 10 g/L 5 15 mM 15 mM 15 mM10 g/L 6 15 mM 30 mM 10 g/L 7 15 mM 10 g/L 8 15 mM 15 mM 10 g/L 9 15 mM10 g/L 10 15 mM 15 mM 10 g/L 11 15 mM 15 mM 10 g/L 12 15 mM 15 mM 10 g/L13 15 mM 15 mM 15 mM 10 g/L 14 15 mM 15 mM 15 mM 10 g/L 15 15 mM 10 g/L16 15 mM 15 mM 10 g/L 17 15 mM 10 g/L 18 15 mM 15 mM 10 g/L 19 15 mM 15mM 15 mM 10 g/L 20 15 mM 15 mM 15 mM 10 g/L 21 15 mM 15 mM 15 mM 10 g/L

The results of the lyophilzation experiments are shown in Table 7 below,

TABLE 7 Lyo Buffer — Excipents — — — 26 Citrate HEPES Glycine HistidineAcetate Tris Phosphate Lysine Histidine Glycine Trehalose Raffinose 1 15mM 10 g/L 2 15 mM 15 mM 10 g/L 3 15 mM 15 mM 10 g/L 4 15 mM 15 mM 10 g/L5 15 mM 15 mM 10 g/L 6 15 mM 30 mM 10 g/L 7 15 mM 10 g/L 8 15 mM 15 mM10 g/L 9 15 mM 10 g/L 10 15 mM 15 mM 10 g/L 11 15 mM 15 mM 10 g/L 12 15mM 15 mM 10 g/L 13 15 mM 15 mM 15 mM 10 g/L 14 15 mM 15 mM 15 mM 10 g/L15 15 mM 10 g/L 16 15 mM 15 mM 10 g/L 17 15 mM 10 g/L 18 15 mM 15 mM 10g/L 19 15 mM 15 mM 15 mM 10 g/L 20 15 mM 15 mM 15 mM 10 g/L 21 15 mM 15mM 15 mM 10 g/L

As shown above, either a Citrate or HEPES buffer in combination with anamino acid provided the clearest solution.

In order to assess stability of the reconstituted lyophilized rVWF,VWF:Ag and VWF:RCo tests were performed. VWF:Ag corresponds to theamount of VWF which can be detected in an VWF-specific ELISA usingpolyclonal anti-VWF antibody, while VWF:RCo corresponds to the amount ofVWF which causes agglutination of stabilized platelets in the presenceof ristocetin. Samples were stored at 40° C. Assuming applicability ofthe Arrhenius equation, one month stability at 40° C. is equivalent toapproximately one year at 4° C. The results of the stability experimentsare shown in Table 8 and Table 9 below.

TABLE 8 rVWF: Ag Weeks at 40° formulation 0 4 5 8 1 121.1 89.8 113.0106.6 2 121.8 102.0 114.0 112.8 3 119.9 102.0 105.,0 112.7 4 117.3 100.0108.0 114.4 5 121.2 98.2 117.0 114.9 6 123.8 96.6 107.0 — 7 135.2 96.6112.0 112.4 8 130.6 82.2 108.0 115.7 9 112.0 89.5 109.0 107.0 10 122.487.1 106.0 107.7 11 119.3 97.5 115.0 114.2 12 124.2 109.0 109.0 103.4 13110.2 92.3 106.0 112.4 14 108.9 107.0 103.0 109.0

TABLE 9 rVWF: RCo Weeks at 40° formulation 0 4 5 8 1 86 102 97.0 93.0 284 97 88.0 89.0 3 85 100 87.0 93.0 4 102 81.0 98.0 5 85 89 88.0 98.0 683 102 88.0 7 92 97.0 95.0 8 88 94 90.0 104.0 9 93 91 97.0 100.0 10 9587 87.0 87.0 11 86 93 89.0 99.0 12 84 91 89.0 95.0 13 88 87 96.0 89.0 1490 91 86.0 92.0

The standard deviation for the ELISA is in the range of 10-20%. Theresults above indicate that all of the formulations tested provide goodstability over 8 weeks at 40° C.

Additional stability experiments were performed where different aminoacids were used in the formulations (e.g., glycine, lysine or histidineat 15 mM or 20 m), and where the citrate buffer was varied (e.g., 15, 20or 25 mM). As described above, stability of rVWF was monitored using theVWF:RCo activity assay. Even after 13 months no significant differenceswere observed for VWF:RCo activity values of rVWF stability samplesstored at 40° C. the significance of the measurements were tested with at-Test. The intermediate precision of the assay was determined bycalculating the Coefficient of Variance. In all series of the stabilitydata the CV was below 20% and met the validation criteria of a CV<20%.Based on the above, it can be concluded that rVWF is stable in allcitrate buffer systems tested, independent of buffer molarity and aminoacids added. NWT remains stable for at least 13 months even when storedat 40° C. The potency determination using the VWF:RCo activity assayshows good intermediate precision with CV values below 20%.

Thus, in view of the data presented herein, a formulation was proposedfor rVWF including 15 mM citrate (Na₃Citrate×2H₂O), 15 mM glycine, 10g/L, Trehalose, 20 g/L Mannitol, 0.1 g/L TWEEN-80®, pH 7.3.

EXAMPLE 4 Long Term Stability

Accelerated and Long-Term Stability Testing

Studies were conducted to evaluate the stability of the rVWF final drugproduct (FDP) stored at both the recommended and elevated storageconditions. Data from the elevated storage conditions provides assurancethat deviations in the temperature will not impact the quality of therVWF FDP and will be used to extrapolate the acceptable expiry conditionof the material in the absence of real-time, real-condition stabilitydata.

The current specification is ≤3.0% residual moisture (as determinedusing the Karl Fischer Method). Lots rVWFF#4FC, rVWFF#5FC, rVWFF#6FC andrVWFF#7FC were released with moisture levels of 1.2%, 1.3%, 1.2%, and1.5% respectively. Based on the past experience with other products withsimilar vial and stopper configurations, it is expected that any rVWFlots released with approximately 1.3% residual moisture will meet thespecification limit of ≤3.0% at the end of the proposed shelf life (i.e.24 months at the intended storage temperature of 5° C.±3° C.).

Long-term stability studies at the recommended storage condition (i.e.5° C.±3° C.) and elevated temperatures (i.e. 40° C.±2° C.) wereconducted with four rVWF FDP lots that have been manufactured. Thesestudies have provided sufficient data to compare the stability behaviorof the individual clinical lots.

The stability protocol, including a description of thestability-indicating assays and stability-acceptance criteria, can befound in Table 10 which also contains information related to the rVWFFDP lots evaluated in the stability studies.

TABLE 10 Storage Conditions Completed (and Proposed) (° C.) Batch NumberTest Intervals 5° C. ± 3° C. rVWF#1FC 0, 1, 2, 3, 6, 9, 12, 18, 24months 30° C. rVWF#1FC 0, 1, 3, 6 months 5° C. ± 3° C. rVWF#2FC 0, 1, 2,3, 6, months 5° C. ± 3° C. rVWF#3FC 0, 1, 2, 3, 6, 9, 12, 18, 24 months30° C. rVWF#3FC 0, 0.5, 1, 2, 3, 6 months 40° C. rVWF#3FC 0, 0.5, 1, 2,3 months 5° C. ± 3° C. rVWF#4FC 0, 1, 2, 3, 6, 9, 12, 18, 24, (30)months 40° C. rVWF#4FC 0, 1, 2, 3, 6, 9 months 5° C. ± 3° C. rVWF#5FC 0,1, 2, 3, 6, 9, 12, 18, (24, 30) months 40° C. rVWF#5FC 0, 1, 2, 3 6, 9months 5° C. ± 3° C. rVWF#6FC 0, 1, 2, 3, 6, 9, 12, (18, 24, 30) months40° C. rVWF#6FC 0, 1, 2, 3, 6, 9 months 5° C. ± 3° C. rVWF#7FC 0, 1, 2,3, 6, 9, 12, (18, 24, 30) months 40° C. rVWF#7FC 0, 1, 2, 3, 6, 9 months

Summary and Discussion of Overall Stability (24 Months)

The rVWF FDP stability data presented is comprised of the following:

1. 24 months data of long-term studies at 5° C.±3° C. (complete testing)and 6 months intermediate data at 30° C.±2° C. (complete testing) forlot rVWF#1FC;

2. 6 months data at 5° C.±3° C. (complete testing) for lot rVWF#2FC;

3. 24 months data of long-term studies at 2-8° (complete testing), 6months data at 30° C.±2° C. and 3 months data at 40° C.±2° C. (completetesting) for lot rVWF#3FC;

4. 24 months stability data at 5° C.±3° C. and 9 months data at 40°C.±2° C. for lot rVWFF#4FC;

5. 24 months stability data at 5° C.±3° C. and 9 months data at 40°C.±2° C. for lot rVWFF#5FC;

6. 12 months stability data at 5° C.±3° C. and 9 months data at 40°C.±2° C. for lot rVWFF#6FC; and

7. 12 months stability data at 5° C.±3° C. and 9 months data at 40°C.±2° C. for lot rVWFF#7FC

The variation observed in residual moisture for lots rVWFF#4FC,rVWFF#5FC, rVWFF#6FC and rVWFF#7FC has remained well below theacceptance criterion 3%, and has not impacted the functional activity(VWF:RCo). There was no observable change in the stability results forqualitative analytical techniques (i.e. appearance, SDS-PAGE analysis,etc.) for the lots manufactured to be suitable for use in thenon-clinical and clinical studies. Similarly, there was no trend indecreasing stability for the total protein analysis, the VWF:Ag analysisor the observed number of VWF multimers during storage.

Variation in both the ratio of VWF:RCo activity to VWF:Ag activity andthe VWF:RCo data presented for lots rVWF#1FC, rVWF#2FC and rVWF#3FC waslikely the result of variation of the test method, the fact that theindividual VWF:RCo stability test results consisted of data from asingle determination of one stability sample, and/or data from thenon-Ph. Eur.-conforming method assay methodology. All testing timepoints for the non-clinical lots subsequent to the modification of theassay methodology to the Ph.Eur.-conforming assay were tested using boththe original and new assay methodology.

The rVWF FDP manufactured at a large-scale exhibited similar stabilitycharacteristics to the rVWF FDP lots manufactured at an experimentalscale. These rVWF FDP lots maintained VWF:RCo activity for up to 24months of storage at 5° C.±3° C. There was no change in the VWF multimerpattern in samples of the large-scale lots currently on stability, evenafter 6 months of storage at 30° C.±2° C. or 9 months storage at 40°C.±2° C. Table 11 shows results for VWF:RCo, VWF:Ag and VWF multimerpattern of the batches rVWF#4FC, rVWF#5FC, rVWF#6FC and rVWF#7FC storedunder stress condition at 40° C.±2° C. The results indicate stability atelevated temperature storage conditions for 9 months which can beextrapolated into a shelf life of more than 3 years at ambienttemperatures or even more under refrigerated conditions.

TABLE 11 Results at Time (Months) Attribute Specification 0 1 2 3 6 9Stability Data for rVWF#4FC at 40° C. ± 2° C. VWF:RCo 70-150 130 117 118127 132 142 Activity [U/ml]¹⁾ VWF:Ag Report result 86 87 79 81 79 86ELISA (U/ml) VWF multimer Report result 21 20 20 20 21 18 analysisStability Data for rVWF#5FC at 40° C. ± 2° C. VWF:RCo 70-150 107 119 120116 132 134 Activity [U/ml]¹⁾ VWF:Ag Report result 94 86 84 91 90 79ELISA (U/ml) VWF multimer Report result 20 20 18 19 20 19 analysisStability Data for rVWF#6FC at 40° C. ± 2° C. VWF:RCo 70-150 118 111 126129 130 119 Activity [U/ml]¹⁾ VWF:Ag Report result 85 95 86.3 73.5 80.870.3 ELISA (U/ml) VWF multimer Report result 20 19 20 20 20 n.t.analysis Stability Data for rVWF#7FC at 40° C. ± 2° C. VWF:RCo 70-150111 115 122 105 99 112 Activity [U/ml]¹⁾ VWF:Ag Report result 87.3 85.377.5 68.8 75 73.8 ELISA (U/ml) VWF multimer Report result 21 20 20 19 1919 analysis

An analysis of covariance (ANCOVA analysis) demonstrated that thedifference in slopes of the regression lines (lots rVWFF#4FC, rVWFF#5FC,rVWFF#6FC and rVWFF#7FC stored at 5° C.±3° C.) is not significant(p=0.906), allowing the VWF:RCo activity data to be pooled as describedin ICH Q1A (R2). The difference in elevation of the trend lines of theindividual lots is also not significant. Extrapolation of the pooledworse case slope, as shown in FIG. 1, shows that the confidenceintervals are well within the acceptance criteria for a minimum of 24months. The lower confidence interval for the mean curve decreases to80% of initial activity at 51 months (80% is also the maximum differencebetween estimated potency and stated potency for Human von WillebrandFactor in Ph.Eur). The pooled worse case slope shows a decrease of0.0344 U VWF:RCo per month. This comparison shows that stabilitycharacteristics of the rVWF FDP, specifically the VWF:RCo activity, didnot change as a result of the changes in the production process. Theabove extrapolation supports the extension of the provisional shelf lifeof NWT FDP to 24 months when stored at the recommended storagetemperature.

The transfer of moisture from the stopper to the lyophilized product isdependent on the stopper material and is influenced by the residualmoisture of the stopper after sterilization, the humidity at which thesample is stored and the intrinsic moisture transfer rate of thestopper. The residual moisture in the lots rVWFF#4FC, rVWFF#5FC,rVWFF#6FC and rVWFF#7FC stored at 5° C.±3° C. was comparable (thedifference in comparison of slopes being not significant, with p=0.734),as shown in FIG. 2. Lots stored at the elevated temperature condition40° C.±2° C. also showed a comparable increase in residual moisture over9 months (FIG. 3). ANCOVA analysis demonstrates here that the differencein slope of the regression lines is comparable (p=0.546). FIG. 3 showsthe extrapolation of the worse case pooled slope up to 24 months.

These are sufficient data to support the use of lots rVWFF#6FC andrVWFF#7FC for the duration of the described expiry period of 24 monthswhen stored at 5° C.±3° C.

Proposed Storage Conditions and Shelf Life

The recommended storage condition for the rVWF FDP is 5° C.±3° C. Aprovisional shelf life of 24 months for the rVWF FDP is thereforeproposed when stored at the recommended storage condition. The shelflife for the rVWF FDP lots likely can be further extended based onadditional data to be generated for longer storage periods.

What is claimed is:
 1. A stable lyophilized pharmaceutical formulationof a recombinant von Willebrand Factor (rVWF) comprising: (a) a rVWF;(b) one or more buffering agents; (c) one or more amino acids; (d) oneor more stabilizing agents; and (e) one or more surfactants; whereinsaid rVWF comprises a polypeptide having the amino acid sequence set outin SEQ ID NO: 3 and encoded by a polynucleotide that hybridizes to thepolynucleotide set out in SEQ ID NO: 1 under moderately stringenthybridization conditions, wherein the polypeptide causes agglutinationof stabilized platelets in the presence of ristocetin, or of binding toFactor VIII; wherein said buffer comprises a pH buffering agent selectedfrom the group consisting of citrate and HEPES at 15 mM and said pH isin a range of about 2.0 to about 12.0; said amino acid is selected fromthe group consisting of glycine, lysine, and histidine at aconcentration of about 1 to about 500 mM; said stabilizing agent is at aconcentration of about 0.1 to about 1000 mM and is selected from thegroup consisting of mannitol, lactose, sorbitol, xylitol, sucrose,trehalose, mannose, maltose, glucose, raffinose, cellobiose,gentiobiose, isomaltose, arabinose, glucosamine, fructose andcombinations of these stabilizing agents; and said surfactant is at aconcentration of about 0.01 g/L to about 0.5 g/L.
 2. The formulation ofclaim 1 wherein the buffering agent is citrate.
 3. The formulation ofclaim 1 wherein pH is in the range of about 6.0 to about 8.0.
 4. Theformulation of claim 3 wherein pH is in the range of about 6.5 to about7.5.
 5. The formulation of claim 3 wherein the pH is about 7.3.
 6. Theformulation of claim 1 wherein the buffering agent is citrate and the pHis about 7.3.
 7. The formulation of claim 1 wherein the amino acid is ata concentration range of about 1 mM to about 300 mM.
 8. The formulationof claim 7 wherein the amino acid is glycine at a concentration of about15 mM.
 9. The formulation of claim 1 wherein the buffering agent iscitrate and the pH is about 7.3; and wherein the amino acid is glycineat a concentration of about 15 mM.
 10. The formulation of claim 1wherein the stabilizing agents are trehalose at a concentration of about10 g/L and mannitol at a concentration of about 20 g/L.
 11. Theformulation of claim 1 wherein the surfactant is selected from the groupconsisting of digitonin, Triton X-100, Triton X-114, polysorbate 20,polysorbate 80 and combinations of these surfactants.
 12. Theformulation of claim 11 wherein the surfactant is polysorbate 80 atabout 0.01 g/L.
 13. The formulation of claim 1 wherein the bufferingagent is citrate at a concentration of about 15 mM at about pH 7.3;wherein the amino acid is glycine at a concentration of about 15 mM;wherein the stabilizing agents are trehalose at a concentration of about10 g/L and mannitol at a concentration of about 20 g/L; and wherein thesurfactant is polysorbate 80 at about 0.1 g/L.
 14. A stable lyophilizedpharmaceutical formulation of a recombinant von Willebrand Factor (rVWF)comprising: (a) a rVWF; (b) one or more buffering agents; (c) one ormore amino acids; (d) one or more stabilizing agents; and (e) one ormore surfactants; wherein the formulation is prepared by lyophilizing asolution comprising: (a) said rVWF comprising a polypeptide having theamino acid sequence set out in SEQ ID NO: 3 and encoded by apolynucleotide that hybridizes to the polynucleotide set out in SEQ IDNO:1 under moderately stringent hybridization conditions; (b) saidbuffer comprising a pH buffering agent in a range of about 0.1 mM toabout 500 mM and having a pH in a range of about 2.0 to about 12.0;wherein the buffering agent is citrate; (c) said amino acid at aconcentration of about 1 to about 500 mM; wherein the amino acid isglycine; (d) said stabilizing agent at a concentration of about 0.1 toabout 1000 mM; wherein the one or more stabilizing agents is mannitoland trehalose; and (e) said surfactant at a concentration of about 0.01g/L to about 0.5 g/L; wherein the surfactant is polysorbate
 80. 15. Astable lyophilized pharmaceutical formulation of a recombinant vonWillebrand Factor (rVWF) comprising: (a) a rVWF; (b) one or morebuffering agents; (c) one or more amino acids; (d) one or morestabilizing agents; and (e) one or more surfactants; wherein theformulation is prepared by lyophilizing a solution comprising: (a) saidrVWF comprising a polypeptide having the amino acid sequence set out inSEQ ID NO: 3 and encoded by the polynucleotide that hybridizes to thepolynucleotide set out in SEQ ID NO:1 under moderately stringenthybridization conditions; (b) said buffer comprising a pH bufferingagent in a range of about 0.1 mM to about 500 mM and having a pH in arange of about 6.5 to about 7.5; wherein the buffering agent is citrate;(c) said amino acid at a concentration of about 1 to about 500 mM;wherein the amino acid is glycine; (d) said stabilizing agent at aconcentration of about 0.1 to about 1000 mM; wherein the one or morestabilizing agents is mannitol and trehalose; and (e) said surfactant ata concentration of about 0.01 g/L to about 0.5 g/L, wherein thesurfactant is polysorbate 80.